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D C Wathes, G E Mann, J H Payne, P R Riley, K R Stevenson, and G E Lamming

Abstract

The regulation of oxytocin, oestradiol and progesterone receptors in different uterine cell types was studied in ovariectomized ewes. Animals were pretreated with a progestogen sponge for 10 days followed by 2 days of high-dose oestradiol to simulate oestrus. They then received either low-dose oestradiol (Group E), low-dose oestradiol plus progesterone (Group P) or low-dose oestradiol, progesterone and oxytocin (via osmotic minipump; Group OT). Animals (three to six per time-point) were killed following ovariectomy (Group OVX), at oestrus (Group O) or following 8, 10, 12 or 14 days of E, P or OT treatment. In a final group, oxytocin was withdrawn on day 12 and ewes were killed on day 14 (Group OTW). Oxytocin receptor concentrations and localization in the endometrium and myometrium were measured by radioreceptor assay, in situ hybridization and autoradiography with the iodinated oxytocin receptor antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2 9]-vasotocin. Oestradiol and progesterone receptors were localized by immunocytochemistry.

Oxytocin receptors were present in the luminal epithelium and superficial glands of ovariectomized ewes. In Group O, endometrial oxytocin receptor concentrations were high (1346 ± 379 fmol [3H]oxytocin bound mg protein−1) and receptors were also located in the deep glands and caruncular stroma in a pattern resembling that found at natural oestrus. Continuing low-dose oestradiol was unable to sustain high endometrial oxytocin receptor concentrations with values decreasing significantly to 140 ± 20 fmol mg protein−1 (P<0·01), localized to the luminal epithelium and caruncular stroma but not the glands. Progesterone treatment initially abolished all oxytocin receptors with none present on days 8 or 10. They reappeared in the luminal epithelium only between days 12 and 14 to give an overall concentration of 306 ± 50 fmol mg protein−1. Oxytocin treatment caused a small increase in oxytocin receptor concentration in the luminal epithelium on days 8 and 10 (20 ± 4 in Group P and 107 ± 35 fmol mg protein−1 in Group OT, P<0·01) but the rise on day 14 was not affected (267 ± 82 in Group OT and 411 ± 120 fmol mg protein−1 in Group OTW). In contrast, oestradiol treatment was able to sustain myometrial oxytocin receptors (635 ± 277 fmol mg protein−1 in Group O and 255 ± 36 in Group E) and there was no increase over time in Groups P, OT and OTW with values of 61 ± 18, 88 ± 53 and 114 ± 76 fmol mg protein−1 respectively (combined values for days 8–14). Oestradiol receptor concentrations were high in all uterine regions in Group O. This pattern and concentration was maintained in Group E. In all progesterone-treated ewes, oestradiol receptor concentrations were lower in all regions at all time-points. The only time-related change occurred in the luminal epithelium in which oestradiol receptors were undetectable on day 8 but developed by day 10 of progesterone treatment. Progesterone receptors were present at moderate concentrations in the deep glands, caruncular stroma, deep stroma and myometrium in Group O. Oestradiol increased progesterone receptors in the luminal epithelium, superficial glands, deep stroma and myometrium. Progesterone caused the loss of its own receptor from the luminal epithelium and superficial glands and decreased its receptor concentration in the deep stroma and myometrium at all time-points. There was a time-related loss of progesterone receptors from the deep glands of progesterone-treated ewes between days 8 and 14.

These results show differences in the regulation of receptors between uterine regions. In particular, loss of the negative inhibition by progesterone on the oxytocin receptor by day 14 occurred only in the luminal epithelium, but is unlikely to be a direct effect of progesterone as no progesterone receptors were present on luminal epithelial cells between days 8 and 14. The presence of oxytocin receptors in the luminal epithelium of ovariectomized ewes suggests that oestradiol is not essential for oxytocin receptor synthesis at this site. Oestradiol was able to sustain its own receptor at all sites, but high circulating progesterone was always inhibitory to oestradiol receptors. In general, oestradiol stimulated progesterone receptors in epithelial cells whereas progesterone abolished its own receptor from epithelial cells over a period of time, but had a lesser effect on stromal cells. The concentration of all three receptors is therefore differentially regulated between different uterine cell types, suggesting the importance of paracrine effects which remain to be elucidated.

Journal of Endocrinology (1996) 151, 375–393

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T. S. JUSS and J. B. WAKERLEY

Experiments were performed on anaesthetized lactating rats to investigate the effects of radiofrequency lesions of the mesencephalon on the milk-ejection reflex. In lesioned and control rats, intramammary pressure recordings were used to estimate oxytocin release (number and relative amplitude of the intermittent milk-ejection responses) during a 3-h suckling test with ten pups. Bilateral lesions (diameter 0·5–1·5 mm) of the lateral tegmentum (near the brachium of the inferior colliculus and medial geniculate body) seriously disrupted the milk-ejection reflex, reducing the number of rats ejecting milk (two out of ten v. all 12 controls, P<0·001) and the amount of oxytocin they released (1·35±0·35 (s.e.m.) v. 15·52±2·19 mu. for controls, P<0·05). Unilateral lesions of the lateral tegmentum also impaired milk ejection and, if the suckling stimulus was restricted only to the contralateral nipples, oxytocin release was virtually abolished. Bilateral lesions placed more medially in the intermediate tegmentum were far less disruptive (eight out of nine rats ejected milk), though the amount of oxytocin released in this group (8·64±1·88 mu.) was still significantly (P<0·05) lower than controls. All rats with lesions of the central grey (nine) or ventral tegmentum (eight) displayed reflex milk ejection, as did those with multiple lesions of the tectum, central grey and ventral tegmentum (seven); in these three groups the amounts of oxytocin released (13·88±2·68, 13·10±1·90 and 11·04±1·95 mu. respectively) did not differ significantly from controls. Damage to the ventral tegmentum produced an irregular pattern of milk ejection characterized by occasional abnormally short (<2 min) milk-ejection intervals, though the overall number of responses in 3 h was less than that of controls (20·83±1·82 v. 14·50±1·30 mu., P<0·05). In conclusion, these results delineate two mesencephalic areas of particular importance in the milk-ejection reflex: (a) the lateral tegmentum, which appears to be concerned with transmission of the suckling stimulus from the contralateral nipples and is indispensable for oxytocin release, and (b) the ventral tegmentum which, although not an essential component of the reflex, may contribute to the timing of the intermittent milk-ejection responses.

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W. H. SAWYER and M. MANNING

SUMMARY

The 4-threonine analogues of oxytocin and of mesotocin and isotocin were prepared by solid-phase synthesis. [4-Threonine]-oxytocin is about twice as active as oxytocin in rat uterus assays in vitro and in vivo and about three times as active in fowl vasodepressor assays. It is slightly more active than oxytocin in rat or rabbit milk-ejection assays. When infused intravenously into water-loaded rats it causes much less depression of diuresis than does an equal dose of oxytocin. [4-Threonine]-oxytocin has much less vasopressor activity than oxytocin. [4-Threonine]-mesotocin also shows enhanced oxytocin-like properties. Its oxytocic activity is equal to or greater than that of oxytocin and its fowl vasodepressor potency is about the same as that of [4-threonine]-oxytocin, 1500 u./mg. It also has less antidiuretic and vasopressor activities than mesotocin. Thus 4-threonine analogues, containing nothing but common l-amino acids, appear to have more of the specific oxytocin-like properties and less of the vasopressin-like properties than do oxytocin or mesotocin. Thus they may be considered improvements on the natural hormones. In this respect they are unique among the reported synthetic analogues of natural peptide hormones.

Substitution of 4-threonine in the weakly-active analogue [3-leucine]-oxytocin also increases its oxytocic and fowl vasodepressor activities. Thus a threonine in the 4-position appears to endow oxytocin-like peptides with greater specific activities than do the amino acids that occur naturally in this position, glutamine and serine. These observations may be of interest when considered (a) from an evolutionary viewpoint, (b) in attempting to interpret relations between molecular structures and biological activities, and (c) as describing peptides with more of the desired properties of oxytocin and less of the undesired properties which might have therapeutic advantages over the natural hormone.

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V. J. Ayad, T. J. Parkinson, E. L. Matthews, and M. L. Wild

ABSTRACT

Previous autoradiographic studies have suggested that the regulation of oxytocin receptors differs between endometrial cell types during the ovine oestrous cycle, and that those present on luminal epithelial cells are of particular importance to the regulation of prostaglandin F release during luteal regression. The present autoradiographic study compares the distribution of the endometrial oxytocin receptor in day-15 non-pregnant and pregnant ewes. The distribution of the endometrial oxytocin receptor in day-15 non-pregnant ewes infused with systemic or intrauterine oxytocin has also been investigated.

Continuous, s.c. infusion of oxytocin (150 mmol/24 h) into ewes (n = 6) between days 10 and 15 of the oestrous cycle significantly increased plasma oxytocin concentrations (to approximately 100 pmol/l). There was no similar increase in systemic oxytocin concentrations in ewes receiving intrauterine (i.u.) oxytocin infusions (10 nmol/24 h) between days 10 and 15 of the oestrous cycle (n = 6). Luteolysis was inhibited in all six animals infused with oxytocin (s.c.) and endometrial oxytocin receptor concentrations were significantly lower on day 15 in these animals (12·8 ± 6·5 (s.e.m.) fmol/mg protein; P<0·001) and in pregnant ewes (18·4 ± 15·4 fmol/mg protein; P <0·001; n = 8) than in ewes infused with saline (248·6±67·1 fmol/mg protein; n = 6). While the 125I-labelled oxytocin receptor antagonist, [1-(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2, Thr4, Orn8, Tyr9-NH2]-vasotocin (125I-labelled OTA) clearly labelled glandular epithelia, luminal epithelium and caruncular stromal cells specifically on day 15 in saline (s.c.)-infused ewes, such specific labelling appeared to be reduced or absent from pregnant animals and those infused with oxytocin (s.c.). A significant reduction in the density of labelling of caruncular stroma (P < 0·05) and luminal epithelium (P < 0·001) was confirmed using quantitative densitometric analysis. The reduction in the labelling of endometrium in oxytocin-infused ewes was not caused by the binding of exogenous oxytocin to endometrial binding sites. Oxytocin infusion (i.u.) did not inhibit luteolysis, nor was there any significant difference in the endometrial oxytocin receptor concentration in this group of ewes on day 15 compared with those infused with saline (i.u.). There was also clear specific labelling of luminal epithelial cells with 125I-labelled OTA in ewes receiving oxytocin infused i.u. and quantification of autoradiograms failed to differentiate between these animals and those infused with saline (i.u.).

It was concluded that systemic oxytocin infusion and the early establishment of pregnancy led to a clear reduction in the endometrial oxytocin receptor concentration on luminal epithelial cells, glandular epithelial cells and caruncular stromal cells, but that i.u. oxytocin infusions did not affect any of these receptor populations and notably not the luminal epithelial oxytocin receptor. The results support the contention that the luminal epithelial oxytocin receptor is involved in the luteolytic process.

Journal of Endocrinology (1993) 137, 423–431

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G. J. S. Tan, R. Tweedale, and J. S. G. Biggs

The effects of oxytocin on dispersed luteal cells from human corpora lutea of the menstrual cycle were studied. Oxytocin at a concentration of 4 mi.u./ml produced a slight increase in basal progesterone production. However, higher oxytocin concentrations (400 and 800 mi.u./ml) markedly inhibited both basal and human chorionic gonadotrophin-induced progesterone production. These data provide evidence for an effect of oxytocin on the human corpus luteum. In view of the inhibitory action of oxytocin, increased secretion of this hormone may be important in the demise of the corpus luteum at the end of the menstrual cycle.

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H. DATTA and R. R. CHAUDHURY

Datta & Chaudhury (1968) have shown that [Asp4]-oxytocin, [Ile8]-oxytocin and [Ser4]-oxytocin block the antidiuretic effect of antidiuretic hormone (ADH) when administered 10 min. before it. Further investigations on the mechanism of this blocking effect of [Asp4]-oxytocin are reported in this communication. The first series of experiments were carried out to see if [Asp4]-oxytocin could also block the vasopressor effect of ADH. Adult male rats (250–300 g.) were anaesthetized with urethane (1.4 g./kg.) and the blood pressure recorded on a smoked drum through a cannula in the carotid artery. The external jugular vein was cannulated for administration of the drugs. ADH (arginine vasopressin) was administered at a dose ranging from 0·25 to 4·0 m−u. before and 10 min. after administration of [Asp4]-oxytocin in eight animals. The same dose of ADH was administered before and after [Asp4]-oxytocin which

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M.C. Lacroix, G. Charpigny, and P. Reinaud

ABSTRACT

This study describes the presence in and production by the ovine conceptus of an oxytocin-like peptide during the early stages of development. Oxycotin was measured by radioimmunoassay in ovine conceptuses from days 14 to 30 of pregnancy. Tissue concentrations of oxytocin increased from day 14 (24.8 ± 5 pg/100 mg) until day 19 (122.9 ± 52 pg/100 mg) and then decreased (3 ± 1 pg /100 mg). Oxytocin was released into culture medium by day-15 ovine conceptuses at a rate of 262 ± 55 pg/24 h. Reverse-phase high-performance liquid chromatography (HPLC) analysis of peptides extracted from a pool of ovine conceptuses was conducted using chromatographic conditions developed to separate oxytocin from other nonapeptides. Radioimmunoassay of HPLC fractions for oxytocin revealed an immunoactive conceptus peptide in a single fraction at the same retention time as chromatographed authentic oxytocin. Radioimmunoassay and chromatographic data therefore suggest that this oxytocin-like peptide is similar, if not identical, to authentic oxytocin. Concentrations of oxytocin in conceptus tissue were maximal during the period of inhibition of luteal regression (days 14-19). It is proposed that conceptus oxytocin is involved in the maintenance of luteal function in early pregnancy.

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M. R. Luck and B. Jungclas

ABSTRACT

The effects of catecholamines and ascorbic acid on cultured bovine granulosa cells have been examined to assess their possible role in the initiation and maintenance of luteal oxytocin secretion. The actions of these agents have also been compared with the previously reported ability of follicular theca tissue to enhance oxytocin secretion.

Using granulosa cells cultured in serum-supplemented medium, we observed a highly significant enhancement of oxytocin secretion in the presence of adrenaline and noradrenaline, particularly over the concentration range 1–10 μmol/l. This effect was accompanied by smaller and less consistent changes in progesterone secretion and did not involve any change in the time-course of oxytocin secretion. Acetylcholine was without effect. Ascorbic acid stimulated oxytocin secretion when used alone over a range of concentrations, but was also able to synergize with adrenaline. Lactic acid was ineffective. The stimulation of oxytocin secretion by adrenaline could be blocked by equimolar propranolol, but the stimulation of progesterone was not blocked. Propranolol had a variable effect on the ability of theca tissue to stimulate oxytocin secretion by granulosa cells but the results also suggested the presence of some β-agonistic activity in the culture medium.

We conclude, first, that catecholamines may be involved in the regulation of ovarian oxytocin secretion, secondly, that ascorbate may regulate oxytocin secretion through its involvement in the biosynthesis of oxytocin but also through interaction with catecholamines and, thirdly, that the stimulatory action of theca tissue probably does not involve the action of β-agonists.

J. Endocr. (1987) 114, 423–430

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S Phaneuf, G Asbóth, M P Carrasco, G N Europe-Finner, F Saji, T Kimura, A Harris, and A López Bernal

Abstract

We have recently provided evidence for the desensitization of oxytocin receptors in human myometrial cells. In the present study, we have investigated the possible mechanisms by which oxytocin (OT) might regulate OT receptor density. The steady state level of OT binding in cultured myometrial cells was 220 × 103 binding sites/cell, but this was time-dependently reduced to 27 × 103 sites/cell by exposure to OT for up to 20 h. Similarly, OT exposure decreased the binding of OT to cell membranes. In contrast, Western blotting data showed that the total amount of OT receptor protein was not affected by OT treatment for up to 48 h. Flow cytometry experiments demonstrated that OT receptors are not internalized during prolonged exposure of the cells to OT. However, RNase protection assays and Northern analysis showed that OT receptor mRNA was reduced by OT treatment to reach a new low steady state level with a time course similar to that of the disappearance of cell surface OT binding sites. Possible mechanisms involved in mRNA down-regulation include transcriptional suppression and destabilization of mRNA by RNA binding proteins.

Journal of Endocrinology (1997) 154, 7–18

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J. B. WAKERLEY, R. E. J. DYBALL, and D. W. LINCOLN

Recordings of intramammary pressure have been used in recent experiments to demonstrate the intermittent pattern of milk ejection (ME) in the rat (Wakerley & Lincoln, 1971; Lincoln, Hill & Wakerley, 1973). This technique provides 'on-line' but indirect evidence of oxytocin release. Other naturally occurring substances (bradykinin, 5-hydroxytryptamine, acetylcholine and vasopressin) can initiate contractions of the mammary gland and thus mimic the effect of oxytocin (Bisset, Clark, Haldar, Harris, Lewis & Rocha e Silva, 1967). The following experiments were designed to identify oxytocin as the active milk-ejecting principle released during suckling.

Bisset, Clark & Haldar (1970) described an in-vivo method of identifying oxytocin using an antagonist, N-carbamyl-O-methyl-oxytocin, which acts as a competitor for oxytocin receptors. The effects of this antagonist on the contractions of the mammary gland during natural ME were examined. Lactating rats were anaesthetized with tribromoethanol (Lincoln et al. 1973) and two mammary glands in the